Grantee Research Project Results
1999 Progress Report: Mechanisms in Toxicological Interactions of Genotoxic Teratogens in Mixture with DNA
EPA Grant Number: R825809Title: Mechanisms in Toxicological Interactions of Genotoxic Teratogens in Mixture with DNA
Investigators: Shank, Ronald C. , Said, Boctor
Institution: University of California - Irvine
EPA Project Officer: Aja, Hayley
Project Period: October 1, 1997 through September 30, 1999 (Extended to September 30, 2001)
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
Project Amount: $505,497
RFA: Issues in Human Health Risk Assessment (1997) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
The study is conducting experiments to investigate the mechanisms by which the presence of: (1) a single modified guanine in a polynucleotide alters the formation of a second guanine adduct at another site in the oligomer, and (2) modified guanines in genomic DNA alter reaction of a second genotoxin with that DNA. Small and bulky carcinogens are represented by alkylating and arylating agents, respectively.Progress Summary:
Most animal studies on genotoxins focus on exposures to single chemicals; however, humans usually are exposed to mixtures of genotoxins. Developmental toxicity and cancer risks of genotoxins in mixture are generally estimated by assuming additivity of the components. Two or more genotoxins acting sequentially may present a greater or lesser risk than could be predicted by assuming additivity. Previously, we showed in both in vitro and in vivo systems that the presence of an N-acetoxy-acetylaminofluorene (N-AcO-AAF)-guanine adduct, but not an N-aminofluorene adduct, in DNA inhibited the subsequent formation of aflatoxin B1-8,9-epoxide adducts, but not methyl adducts from dimethylsulfate. This finding generated the hypothesis that changes in the conformational state of the DNA helix was the basis for the non-additive effect of these two genotoxins. The study was extended to investigate the changes in DNA conformation under the reaction conditions used in the above studies. Chemical probes were used to confirm that the AAF adduct caused local distortions in the helix. In studies using oligonucleotides, two probes?diethylpyro-carbonate and hydroxylamine?were used to show that there was significant hyperreactivity in the immediate area of Gua-C8-N-AcO-AAF adducts. Both probes sense the degree of denaturation or single strandedness of DNA, and the results support that there is local denaturation around AAF adducts in the test systems used.In an in vivo study, the effect of one genotoxin on the mutagenicity of a second genotoxin was evaluated in a Salmonella typhimurium assay for several pairs of compounds. Pretreatment of frameshift strain TA 98 (DuvrB, +pKM101) or TA 1538 with AFB1-8,9-epoxide (17.3 ng/plate) enhanced the mutagenicity of N-AcO-AAF approximately three times above theoretical additive effects. The same effect was seen in strain TA100 (DuvrB, -pKM101) in which a 2-fold enhancement of N-AcO-AAF mutagenicity was observed. Pretreatment of TA 98 with trans-7,8-diol-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti) (BPDE) (75 ng/plate) enhanced N-AcO-AAF mutagenicity four to six times, and a non-adducting intercalator, ethidium bromide (20 ng/plate), enhanced N-AcO-AAF mutagenicity five times above the theoretical value assuming additivity. Neither of the initial genotoxins, AFB1-8,9-epoxide or BPDE, or the intercalator, ethidium bromide, was mutagenic by itself in TA 98. All of the initial genotoxin treatments followed clear dose-response relationships.
Another study investigated the mutational spectrum in the hisD3052 allele of Salmonella. Colonies treated with the above combinations of genotoxins were screened for the presence of a ?2 deletion of GC or CG within the hot spot target sequence CCGCGCGCGG using a 32P end-labeled probe (5'-CTGCCGCGCGGACACCG-3'). Of the spontaneous mutations, 47?53 percent were ?2 deletions; in cells treated with N-AcO-AAF alone, 87?93 percent of the mutations were ?2 deletions. The frequency of ?2 deletions in bacteria pretreated with AFB1-8,9-epoxide, BPDE, or ethidium bromide was 92.5 percent, 91 percent, and 95 percent, respectively. Pretreatment of the Salmonella with the intercalating genotoxins increased the potency of N-AcO-AAF as a mutagen, but did not change the type of mutation.
To study the effect of N-AcO-AAF on the mutagenicity of AFB1-8,9-epoxide, a base-substitution strain, TA 100, was pretreated with N-AcO-AAF. In a dose-dependent manner, N-AcO-AAF (0.1?1,000 ng/plate) inhibited the induction of revertants by AFB1-8,9-epoxide (43 ng/plate). A concentration of 1 ng/plate N-AcO-AAF reduced the total number of revertants to 44 percent of that induced by AFB1-8,9-epoxide alone. N-AcO-AAF itself induced mutations in this base-substitution strain. Concentration-response relationships for these enhancing or inhibitory effects were demonstrated using increasing concentrations of the first genotoxin during pretreatment. These results demonstrate effects, other than additive, of sequential exposures to two genotoxins on the induction of mutations in a bacterial system, and suggest that changes in helix conformation induced by bulky agents are important in modulating the formation of subsequent adducts.
Future Activities:
The next series of studies will focus on the mechanism(s) of action by which one genotoxic teratogen/carcinogen modulates the interaction(s) with subsequent genotoxins. Bacterial mutagenicity studies will be expanded to include more strains to test proposed mechanisms of action by mixtures of genotoxins. Changes in DNA helix conformation also will be investigated in greater detail.Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 7 publications | 3 publications in selected types | All 3 journal articles |
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Ross MK, Mathison BH, Said B, Shank RC. 5-Methylcytosine in CpG sites and the reactivity of nearest neighboring guanines toward the carcinogen aflatoxin B1-8,9-epoxide. Biochemical and Biophysical Research Communications 1999;254(1):114-119. |
R825809 (1999) R825809 (2000) R825809 (Final) |
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Ross MK, Said B, Shank RC. DNA-damaging effects of genotoxins in mixture: modulation of covalent binding to DNA. Toxicological Sciences 2000;53(2):224-236. |
R825809 (1998) R825809 (1999) R825809 (2000) R825809 (Final) |
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Said B, Ross MK, Hamade AK, Matsumoto DC, Shank RC. DNA-damaging effects of genotoxins in mixture: nonadditive effects of aflatoxin B1 and N-acetylaminofluorene on their mutagenicity in Salmonella typhimurium. Toxicological Sciences 1999;52(2):226-231. |
R825809 (1999) R825809 (2000) R825809 (Final) |
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Supplemental Keywords:
genotoxic mixtures, teratogens, carcinogens, DNA damage by mixtures., RFA, Health, Scientific Discipline, Waste, Toxicology, Genetics, Environmental Chemistry, Chemistry, chemical mixtures, Risk Assessments, chemical probes, synthetic oligonucleotides, genetic analysis, genotoxic teratogens, human exposure, metabolic activation, DNA, toxic environmental contaminants, toxicodynamics, reproductive health, teratogen mixtures, cancer riskProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.